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S H Chang

Publications and source records attributed to S H Chang.

At least 163 records · Page 9Linked to original sources
PubMed1982

Partial P1 nuclease digestion as a probe of tRNA structure.

Partial P1 nuclease digestion of 5'-32P-labeled tRNAs, followed by polyacrylamide gel electrophoresis and autoradiography, results in a series of bands of unequal intensities. Comparison of the digestion profiles of two conformers of yeast tRNA3Leu shows that P1 nuclease is sensitive to structural features of its substrate. Examination of the digestion pattern of yeast tRNAPhe in light of its known three-dimensional conformation shows that the enzyme's activity reflects the primary, secondary and tertiary levels of tRNA structure.

Base Sequence↗
PubMed1981

The nucleotide sequence of Euglena cytoplasmic phenylalanine transfer RNA. Evidence for possible classifications of Euglena among the animal rather than the plant kingdom.

The nucleotide sequence of cytoplasmic phenylalanine tRNA from Euglena gracilis has been elucidated using procedures described previously for the corresponding chloroplastic tRNA [Cell, 9, 717 (1976)]. The sequence is: pG-C-C-G-A-C-U-U-A-m(2)G-C-U-Cm-A-G-D-D-G-G-G-A-G-A-G-C-m(2)2G-psi-psi-A-G-A-Cm -U-Gm-A-A-Y-A-psi-C-U-A-A-A-G-m(7)G-U-C-*C-C-U-G-G-T-psi-C-G-m(1)A-U-C-C-C-G-G- G-A-G-psi-C-G-G-C-A-C-C-A. Like other tRNA Phes thus far sequenced, this tRNA has a chain length of 76 nucleotides. The sequence of E. gracilis cytoplasmic tRNA Phe is quite different (27 nucleotides out of 76 different) from that of the corresponding chloroplastic tRNA but is surprisingly similar (72 out of 76 nucleotides identical) to that of tRNA Phe from mammalian cytoplasm. This extent of sequence homology even exceeds that found between E. gracilis and wheat germ cytoplasmic tRNA Phe. These findings raise interesting questions on the evolution of tRNAs and the taxonomy of Euglena.

Animals↗
PubMed1981

The bacteriorhodopsin gene.

The bacteriorhodopsin gene has been identified in a 5.3-kilobase restriction endonuclease fragment isolated from Halobacterium halobium DNA, using a cloned cDNA fragment as the probe. Of the 1229 nucleotides whose sequence was determined in the genomic fragment, 786 correspond to the structural gene of bacteriorhodopsin, 360 are upstream from the initiator methionine codon, and 83 are downstream from the COOH terminus. The bacteriorhodopsin gene codes for a precursor sequence of 13 amino acids at the NH2 terminus, 248 amino acids that are present in the mature protein and an additional aspartic acid at the COOH terminus. This determination of the DNA sequence for an archaebacterial gene reveals that the standard genetic code is used; however, there is a marked preference for either G or C in the third codon position. The gene does not contain any intervening sequences and no prokaryotic promoter can be identified in the region immediately upstream from the structural gene. The bacteriorhodopsin mRNA contains at the 5' terminus only three nucleotides beyond the initiating AUG codon and this terminus can form a hairpin structure. Immediately downstream from this structure there is a sequence complementary to the 3' terminus of H. halobium 16S rRNA.

Amino Acid Sequence↗
PubMed1981

Bacteriorhodopsin: partial sequence of mRNA provides amino acid sequence in the precursor region.

mRNA for bacteriorhodopsin from Halobacterium halobium has been partially purified. By using this mRNA as template in the presence of reverse transcriptase RNA-dependent DNA nucleotidyltransferase and a 5'-[32P] synthetic oligodeoxyribonucleotide corresponding to amino acids 9-12 of bacteriorhodopsin as primer, we have isolated the major 5'-[32P]cDNA product, approximately 80 nucleotides long, and determined its sequence. Based on the cDNA sequence, the 5'-proximal sequence of bacteriorhodopsin mRNA is G-C-A-U-G-U-U-G-G-A-G-U-U-A-U-U-G-C-C-A-A-C-A-G-C-A-G-U-G-G-A-G-G-G-G-G-U-A-U-C -G-C-A-G-G-C-C-C-A-G-A-U-C-A-C-C-G-G-A-C-G-U-C-C-G. This includes the expected sequence for amino acids 1-8 and shows that bacteriorhodopsin is synthesized as a precursor that is at least 13 amino acids longer (Met-Leu-Glu-Leu-Leu-Pro-Thr-Ala-Val-Glu-Gly-Val-Ser) at the NH2 terminus. Agarose/urea gel electrophoresis of the partially purified mRNA showed several bands; of these, a major one hybridized with 5'-[32P]cDNA. These results suggest that the bacteriorhodopsin mRNA in the partially purified preparation is homogeneous in size and that it constitutes a substantial portion of the RNA preparation subjected to electrophoresis.

Amino Acid Sequence↗
PubMed1980

The nucleotide sequence of two bovine lens phenylalanine tRNAs. Possible activation of a new phenylalanine tRNA gene during differentiation of lens cells.

Bovine lens fiber cells contain two major isoacceptors of phenylalanine tRNA, tRNA1Phe and tRNA2Phe whereas lens epithelial cells contain mainly tRNA2Phe (Ortwerth, B.J,, Yonuschot, G.R., Heidlege, J.F., Chu-Der, O.M.Y., Juarez, D., and Hedgcoth, C. (1975) Exp. Eye Res. 20, 417-426). Thus, the induction of a new species of tRNAPhe accompanies the differentiation of lens cells in vivo. These two species of tRNAPhe have been purified and their sequences have been analyzed. The results of the analyses show that the sequence of tRNA2Phe is identical with beef liver tRNAPhe while tRNA1Phe differs from tRNA2Phe by a guanosine to adenosine substitution at position 57. Therefore, the T psi CG sequence in the ribosylthymine loop of tRNA2Phe is replaced by T psi CA in tRNA1Phe, This observation demonstrates a case in which a change in sequence of tRNA isoacceptors accompanies cell development rather than changes in the level of post-transcriptional modification as noted in other systems (reviewed by Osterman L.A. (1979) Biochimie (Paris) 61, 323-342). The conversion of guanosine to adenosine in the ribosylthymine loop of lens tRNA1Phe suggests a possible activation of a new tRNAPhe gene during the differentiation of lens cells, although the possibility that this replacement may be catalyzed by a transglycosylase type enzyme has not been ruled out.

Animals↗
PubMed1980

A rapid method of cryofixation of tissues in situ for ultracryomicrotomy.

A device is described for the rapid freezing of tissue in situ by a punch biopsy approach using a specially designed cryogun with a highly thermal conductive specimen holder. The cryogun consists of a sampling device using a double, spring-loaded gun mechanism and a system of cryochambers. Ultrathin freeze-dried sections cut from samples obtained with this cryogun are relatively free of artifacts and have few ice crystals. Organelles are seen by natural contrast when cryosections of approximately 1000 A are observed with a transmission electron microscope or in the transmission mode of a scanning electron microscope. The construction of the cryogun is described along with a method of obtaining improved, ultrafast cryofixation of tissue specimens. The reliability of obtaining x-ray microanalysis measurements of diffusible ions where movement within cell compartments has been retained is discussed.

Basement Membrane↗
PubMed1979

Yeast tRNA Leu UAG. Purification, properties and determination of the nucleotide sequence by radioactive derivative methods.

A second major species of leucine tRNA, tRNA Leu UAG (formerly designated tRNA Leu CUA) was purified from baker's yeast in a three-step procedure entailing BD-cellulose chromatography in the presence and absence of Mg2+ and Sephadex G-100 gel filtration. Results of aminoacylation and partial RNase T1 digestion experiments showed that this tRNA retains a native conformation under conditions that denature yeast tRNA Leu m5CAA (tRNA3 Leu). The primary structure of baker's yeast tRNA Leu UAG was elucidated by application of sensitive radioactive isotope derivative ("postlabeling") methods. Complete RNase T1 and A and partial RNase U2 fragments, prepared from non-radioactive tRNA and 5'-half and 3'-half molecules, were separated by two-dimensional polyethyleneimine-cellulose anion-exchange thin-layer chromatography and isolated by a novel micropreparative procedure affording high yields of these compounds in sufficient purity for subsequent tritium derivative analysis. Base composition and sequence of oligonucleotides were analyzed by tritium derivative methods. Molar ratios of the fragments were determined from the radioactivity of 3H-labeled nucleoside trialcohols in combination with base analysis. 2'-O-Methylated guanosine was characterized using the [gamma-32P]ATP/polynucleotide kinase reaction. The analysis of classical complete and partial RNase digests by the tritium derivative methods yielded the complete nucleotide sequence of the tRNA. A total of about 20 A260 units of the RNA was used for analysis, i.e. considerably less material than required for conventional spectrophotometric analysis. A different sequencing approach, consisting of a combination of "readout sequencing" with tritium sequencing of complete RNase T1 and A fragments, was applied to the 3'-half molecule. The 3'-half molecule was labeled with 32P at its 5' terminus, partially degraded with RNase T1, U2, and Phy1 and with alkali, and subjected to polyacrylamide gel electrophoresis. The sequence was read off the gel on the basis of cleavage patterns and size of the fragments. While the readout procedure provided only the positions of A, U, C, and G residues in the chain, additional information from tritium derivative analysis was utilized to define the positions of the modified nucleosides. The readout sequencing procedure was found to require less than 0.01 A260 unit of RNA and the analysis of the complete fragments about 6 A260 units. Interesting structural features of tRNA Leu UAG are (a) the location of unique, leucine tRNA iso-acceptor-specific sequences next to U-8, a constant nucleotide participating in synthetase recognition, (b) the occurrence of 1-methyladenosine in the T loop, a modification not present in the structurally related tRNA Leu m5CAA, and (c) the unusual presence of an unmodified uridine in the first position of the anticodon, which may be related to the unusual coding properties reported for this tRNA.

Adenine↗
PubMed1979

Differences between transfer RNA methylase activity in human diploid fibroblasts during in vitro and in vivo aging.

The change of the specific activity of S-adenosylmethionine: tRNA methyltransferase in cultures of human diploid fibroblasts at different passages has been measured and compared with that in the same type of cells derived from donors of different ages. Whereas the specific activity of tRNA methylase in the in vitro aged cells was found to decline gradually with increasing passage number of the culture, a different activity--age relationship was observed for this enzyme in cells derived from donors of different ages. The activity of tRNA methylase is high in the fetal cells and drops drastically in the "newborn" cells. After a further 10% decline, the activity of this enzyme reaches a steady low level in the postnatal cells from donors ranging in age from 3 months to 94 years. These findings cast doubt on the validity of the assumption that the results obtained from in vitro aging experiments reflect the biochemistry of aging in vivo. The "fetal" enzyme can methylate the "aged" tRNA but the "aged" enzyme cannot methylate the "fetal" tRNA. The fetal cells contain enzyme activities specific for the formation of m1A, m5C and m1G. These activities are low or deficient in "aged" cells. Control experiments showed that all of these results are due neither to the presence of inhibitor or stimulator in the extract nor to effects related to the population density, sex or growth rate of the culture.

Adult↗
PubMed1979

The role of ion shifts in cell injury.

The present paper reviews studies which utilize x-ray microanalysis to determine intracellular ion shifts following several types of cell injury. New data from our own laboratory on several cell injury systems are discussed. Concentration estimates are made by comparison of data from tissues with a series of standards prepared in 20% albumin followed by cryosectioning. Hemorrhagic shock in rats is followed by rapid changes of ions in both muscle and liver. These include increased levels of sodium and chlorine and decreased levels of potassium which can be correlated with deficits in the energy charge. Measurements made over hepatocellular carcinomas in the mouse, induced by safrole show marked changes in comparison with non-transformed cells. These include striking increases in sodium and chloride and decreases in potassium and phosphorus which may be related to growth control. Studies on ischemia produced by arterial clamping in the rat kidney and the dog heart show somewhat similar changes. Moreover, in these models much interest is directed at early increases of cytoplasmic calcium with decreased mitochondrial calcium levels at later intervals. Following reflow, there is a prominent increase of calcium in the cytosol. These changes in calcium may be related to activation of phospholipases producing permeability changes which may contribute to further ion shifts as well as ultimately to cell death. The paper also comments on the use of cryostat sections for some types of routine pathological analysis.

Animals↗
PubMed1977

A continuous-flow enzyme detector for liquid chromatography.

A detection system has been developed for the selective and sensitive detection of enzymes eluting from a liquid chromatographic column. This system monitors a reaction that the enzyme catalyzes and provides a chemical amplification ranging from 10(4) to 10(5). The detection system consists of a reagent or substrate pump, a post-column reactor packed with non-porous spherical glass beads, and a photometric detector. A linear and selective response to a series of enzymes of clinical importance is demonstrated.

Alcohol Oxidoreductases↗
PubMed1977

Proliferating Brenner tumor.

Proliferating Brenner tumor is rare as there are only 17 previously reported cases. A case of proliferating Brenner tumor is presented with special emphasis on the pathologic and histologic findings. This variant of Brenner tumor is considered to be benign, as no recurrences have developed in any reported patients.

Aged↗
PubMed1976

Detection of ion shifts in proximal tubule cells of the rat kidney using x-ray microanalysis.

The present study represents an attempt to combine scanning transmissions electron microscopy and x-ray microanalysis on thin sections at the intracellular level using kidney tubules in both normal and altered conditions. The results show that semi-quantitative estimates made from X-ray microanalysis can predict the pattern of electrolyte change both in direction and degree; these estimated agree with previously determined chemical analysis of identically treated tissue. Before these promising results can be extended to quantitative intracellular compartment analysis, further work is needed in several areas including preparation of adequate standards, improved ultracryomicrotomy techniques, improvement of contrast of ultrathin frozen sections and standardization in computing background measurements.

Animals↗
PubMed1976

High-performance liquid chromatography of proteins.

Proteins were separated on microparticulate bonded phase steric exclusion and anion-exchange chromatography supports. A post-column enzyme detector was developed which gives a specific and sensitive response for enzymes. The three iso-enzymes of creatine phosphokinase were separated and assayed in 4 min and the five isoenzymes of lactic dehydrogenase in 6 min.

Animals↗
PubMed1976

Studies on the pathogenesis of ischemic cell injury. V. Morphologic changes of the pars convoluta (P1 and P2) of the proximal tubule of rat kidney made ischemic in vitro.

In summary, we have described the time course of changes of mitochondria following ischemia of the kidney proximal tubule. The sequence of morphological changes of matrix as well as inner membrane corresponds well with certain functional or physical parameters such as swelling, respiration, substrate metabolism, acceptor control and P/O ratio. This indicates that morphological parameters can be utilized to predict the functional alterations of mitochondria following ischemia in cells. The significant mitochondrial changes are early loss of granules (15-30 min) and condensation (15 min), swelling (30 min), appearance of fluffy densities (30 min) and flocculent densities (after 60 min), degeneration of cristal structure (240 min) and disintegration of mitochondria as structural units (24 h).

Animals↗